Inhibition of by Extracts and Constituents from Angelica archangelica and Geranium sylvaticum Steinthor Sigurdsson* and Sigmundur Gudbjarnason

Science Institute, University of Iceland, Vatnsmyrarvegur 16, IS-101 Reykjavik, Iceland. Fax: +354525 48 86. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 62c, 689Ð693 (2007); received February 2/March 15, 2007 The aim of this study was to explore the acetylcholinesterase (AChE) inhibition of several Icelandic medicinal herbs. Ethanolic extracts of Angelica archangelica seeds and the aerial parts of Geranium sylvaticum proved effective, with IC50 values of 2.20 mg/ml and 3.56 mg/ml, respectively. The activity of imperatorin and xanthotoxin from A. archangelica was measured. μ Xanthotoxin proved much more potent than imperatorin, with an IC50 value of 155 g/ml (0.72 mm) but that for imperatorin was above 274 μg/ml (1.01 mm). However, furanocoumar- ins seem to have a minor part in the total activity of this extract. Synergistic interaction was observed between the extracts of A. archangelica and G. sylvaticum. Several medicinal herbs (Achillea millefolium, Filipendula ulmaria, Thymus praecox and Matricaria maritima) did not show AChE inhibitory activity. Key words: Acetylcholinesterase, Angelica archangelica, Geranium sylvaticum

Introduction and has been reported to have an inhibitory effect on AChE from mouse brain (Kim et al., 2002). Alzheimer’s disease (AD) is an irreversible and Xanthotoxin (8-methoxypsoralen) is one of the chronic neurodegenerative disorder afflicting major furanocoumarins in A. archangelica. It dif- more than 20 million people worldwide (Kamal et fers from imperatorin only by having a methoxy- al., 2006). It is the most common cause of demen- group replacing the isopentenyloxy group at posi- tia in the elderly (Ellis, 2005). Among its charac- tion 8. It has been reported to inhibit AChE from teristics is the presence of extra-neuronal amyloid Drosophila melanogaster with an IC50 value of deposits and progressive deficits in several neuro- 125 μg/ml, and it has been suggested that this inhi- transmitter systems, of which the is af- bition is responsible for its insecticidal activity fected earliest, associated with a decline in levels (Miyazawa et al., 2004). of (Kamal et al., 2006). Since the dis- Geranium sylvaticum (woodland geranium) is covery of cholinergic deficits in patients suffering widespread in Europe, but is seldom referred to from AD, acetylcholinesterase (AChE) has been as a medicinal herb in the recent literature. It has the main target for the treatment of it (Hodges, not been studied previously with respect to its ace- 2006). The AChE inhibitors , , tylcholinesterase activity. galanthamine and are currently avail- Synergy, that is the observation that a combina- able as drugs for general clinical treatment of AD tion of compounds or extracts is more effective (Akhondzadeh and Abbasi, 2006). than can be expected from the activity of its com- Angelica archangelica has been long and widely ponents, has been documented for various phy- used in folk medicine, and it is one of the most tomedicines (Williamson, 2001). Synergistic effect respected medicinal herbs in Nordic countries, on acetylcholinesterase has been suggested by al- where it was cultivated during the Middle Ages kaloids of Amaryllidaceae plants (Orhan and and exported to other parts of Europe. The most Sener, 2003). The isobole method is the method characteristic secondary metabolites of its fruits of choice for demonstrating synergy (Williamson, are essential oils and furanocoumarins (Newall 2001) and was used in this study. et al., 1996). The purpose of this study was to explore the Imperatorin (8-isopentenyloxypsoralen) is the acetylcholinesterase inhibition of extracts from major furanocoumarin of A. archangelica seeds several medicinal herbs growing in Iceland.

0939Ð5075/2007/0900Ð0689 $ 06.00 ” 2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 690 S. Sigurdsson and S. Gudbjarnason · Inhibition of Acetylcholinesterase

Materials and Methods rates were used for subsequent calculations. Gal- anthamine was used as positive control, and had Plant material and extracts μ an IC50 value of 10.8 m in this assay. Achillea millefolium (yarrow, flowers and leaves In the case of imperatorin and xanthotoxin, up separately), Filipendula ulmaria (meadowsweet), to 2% triton X-100 was added to the buffer to en- Thymus praecox (creeping thyme), Matricaria hance the poor solubility of the furanocoumarins, maritima (scentless chamomille), Angelica archan- particularly of imperatorin. The effect of the gelica (seeds) and Geranium sylvaticum (wood- added triton X-100 on the rate of the enzymatic land geranium) were collected in Iceland. The reaction was negligible, but control samples were plant material was dried and stored at room tem- examined containing the same amount of the de- perature. Subsequently, it was extracted with 45% tergent. ethanol in water (v/v) for a minimum of 10 d. HPLC Furanocoumarins In order to establish the concentration of dis- Imperatorin was precipitated from a 45% etha- solved furanocoumarin in the assay, immediately upon completion of the enzymatic assays, the test nolic extract of A. archangelica, growing in Ice- μ land, by adding water, and recrystallized twice solutions were filtered using 0.45 m syringe filters from diethyl ether until its purity was more than (Schleicher & Schuell) and measured by HPLC, upon appropriate dilution. A HP-1100 instrument 95% (monitored by HPLC). Xanthotoxin was pur- with a quaternary pump was used for the mobile chased from Aldrich. An A. archangelica seed ex- phase. The column was from Pecosphere, con- tract with reduced furanocoumarincontent was taings a C18 stationary phase with a particle size made by evaporating 100 ml of the extract on a of 3 μm (33 ¥ 4.6 mm I. D.). Gradient elution was rotary evaporator until 35 ml remained. The re- from 0Ð8 min from 40Ð60% MeOH in water, de- sulting concentrate was centrifuged for 5 min at tection at 305 and 309 nm at room temperature, 14,000 ¥ g. The supernatant was collected and used with a flow rate of 1.0 ml/min. The signal at in AChE inhibition assays. 305 nm was monitored and compared to standard curves with known amounts of xanthotoxin. The Enzyme assay standard curve was applied to other furanocou- The AChE assay was performed by the modified marins by correcting for different molecular method of Ellman et al. (1961). Mouse brains, ei- weights and the respective extinction coefficients ther fresh or stored at Ð20 ∞C, were homogenized at their maxima at about 300 nm (Lee and Soine, in 5 volumes of buffer [10 mm tris(hydroxy- 1969). methyl)-aminoethane-HCl, pH 7.2, 1 m NaCl, Results 50 mm MgCl2 and 1% triton X-100] and then cen- trifuged for 30 min at 14,000 ¥ g. All extraction 45% Ethanolic extracts steps were carried out on ice or at 4 ∞C. The en- The 45% ethanolic extracts of A. archangelica zyme extract was either subsequently used or and G. sylvaticum inhibited AChE dose-depend- stored at Ð70 ∞C. The assays were carried out in ently, with IC50 values of 2.20 mg/ml and 3.56 mg/ 96-wells microtiter plates. 0.25 ml of sample was diluted with 1.21 ml buffer (50 mm sodium phos- Table I. IC50 values of the inhibition of AChE by ex- phate buffer, pH 8). 70 μl of the resulting mixture tracts from G. sylvaticum and A. archangelica and by 5 were added to 10 μl of enzyme solution and thor- mixtures of the extracts. oughly mixed for 1 min at 37 ∞C, before adding % G. sylvaticum IC [mg/ml] 10 μl of reagent mixture (3.5 mm acetylthiocholine 50 and 7 mm dithiobisnitrobenzoic acid). The final Total G. sylvaticum A. archangelica concentrations of the reagents were thus 0.39 mm 0.00 2.20 0.00 2.20 of acetylthiocholine and 0.78 mm of dithiobisni- 37.79 2.03 0.77 1.26 trobenzoic acid. Upon adding the reagents, the so- 47.68 2.02 0.96 1.06 lution was mixed and incubated at 37 ∞C, and the 64.57 2.10 1.36 0.74 absorbance at 412 nm was measured every 30 s for 78.47 2.04 1.60 0.44 5 min. Typically, the rate was linear between 1 and 84.54 2.22 1.88 0.34 100.00 3.56 3.56 0.00 3 min from the start of the reaction, and those S. Sigurdsson and S. Gudbjarnason · Inhibition of Acetylcholinesterase 691 ml, respectively (Table I). The 45% ethanolic ex- tracts of the aerial parts of A. millefolium, F. ulma- ria, T. praecox and M. maritima showed little inhi- bition (Ͻ 24%) at concentrations of 3.1 mg/ml and higher.

Supernatant of concentrated A. archangelica extract The supernatant of the 45% ethanolic extract of A. archangelica seeds, which had been concen- trated in a rotary evaporator and centrifuged, was assayed. 100 ml had been concentrated to 35 ml and the precipating material removed. The sample was tested in quintuplicate at the concentration 2.88 mg/ml, and inhibited the enzyme by (51.0 ð 4.4)%.

Furanocoumarin content in enzyme assays Fig. 1. Dose-dependent inhibition of AChE by the The furanocoumarin content in an enzyme assay extracts from A. archangelica and G. sylvaticum and a with maximal content of A. archangelica seed ex- mixture containing 84.54% G. sylvaticum extract and tract was measured, after pooling the reaction 15.46% A. archangelica extract. Each point represents μ the average of two separate measurements, each done mixtures from six identical wells (90 l each). The in triplicate, and the standard error of the mean. The total furanocoumarin content was 55.2 μg/ml, con- synergistic activity was prominent above 2 mg/ml. sisting of 23.3 μg/ml of imperatorin, 11.6 μg/ml xanthotoxin, 8.1 μg/ml isoimperatorin, 6.3 μg/ml isopimpinellin and 6.0 μg/ml bergapten. The fura- nocoumarin content of the assay with the super- natant of the evaporated extract was assayed and found to contain 3.8 μg/ml of xanthotoxin, whereas other furanocoumarins were not detect- able.

Furanocoumarins Imperatorin showed no significant inhibition ac- tivity on AChE at the maximal concentration μ Ͼ μ measured, 274 g/ml, i.e.,IC50 274 g/ml. The IC50 value of xanthotoxin was found to be 155 μg/ml.

Synergy of combined extracts The extracts from A. archangelica and G. sylvat- Fig. 2. Isobole curve for the concentrations of extracts icum were mixed together in five different ratios from G. sylvaticum and A. archangelica at 50% inhibi- and assayed. The dose-response curve of the mix- tion of AChE. The points show the concentration of ture containing 84.54% of G. sylvaticum, and those each component at IC50 for the individual extracts and for the five mixtures. of both extracts, are shown in Fig. 1. The IC50 val- ues are shown in Table I. Fig. 2 shows the isobole curve of 50% inhibition of the extracts from A. component at IC50 for different mixtures. If the archangelica and G. sylvaticum. The IC50 value of components do not interact, the quantity of each each component is shown on the axes, and the ad- component at IC50 of the mixtures should appear ditional five points show the concentration of each on the straight line between the IC50 values of the 692 S. Sigurdsson and S. Gudbjarnason · Inhibition of Acetylcholinesterase components (Williamson, 2001). Fig. 2 shows, that ml (0.72 mm). As could be anticipated, this is in the mixtures the IC50 values appear under the somewhat different from the value that has been straight line between the IC50 values of the com- established for AChE from Drosophila melano- ponents. This shows synergy, and that the effect is gaster (Miyazawa et al., 2004), namely 0.58 mm greater than expected from their individual dose- (125 μg/ml). response curves. In all mixtures, the synergy is The concentration of xanthotoxin in the seed ex- μ much more prominent in higher doses (above ca. tract reaction mixture at IC50 is less than 11.6 g/ 2 mg/ml) than in lower doses, as can be seen for ml. As the IC50 value of isolated xanthotoxin is the mixture containing 84.54% of G. sylvaticum 155 μg/ml, it is clear that other compounds in the and 15.46% of A. archangelica in Fig. 1. extract explain most of the activity. The total con- centration of furanocoumarins at IC50 is less than Discussion 55 μg/ml Ð more than 40% of which is impera- torin. Thus, if furanocoumarins were the reason Acetylcholinesterase inhibition activity was for the overall activity of the extract, the remain- demonstrated for extracts of A. archangelica and ing furanocoumarins would have to be much more G. sylvaticum. Other plant materials tested, of A. active than xanthotoxin. millefolium, F. ulmaria, T. praecox and M. mari- Another experiment designed to answer the tima, had no significant inhibitory activity. question if furanocoumarins were the main active AChE inhibition has previously been ascribed components was to test the extract from which to furanocoumarins (Kim et al., 2002; Miyazawa most of the furanocoumarins had been removed. et al., 2004). Therefore, it was safe to assume, that The furanocoumarins in question have very lim- the inhibition of the A. archangelica seed extract ited solubility in water, and precipitate when the was due to their presence. The two most abundant ethanol content is reduced considerably. When furanocoumarins in the reaction mixture when the 65% of its volume was evaporated, most or all of extract was assayed were imperatorin and xantho- the ethanol had been removed, and only a small toxin. proportion of xanthotoxin remained in the mix- Imperatorin is the most abundant furanocouma- ture. However, in a sample containing only 3.8 μg/ rin in the extract, comprising 40Ð50% of its total ml of furanocoumarins, all of which was xantho- furanocoumarin content (results not shown). As it toxin, in the assay about 50% inhibition was at- is very poorly soluble in water, 2% triton X-100 tained. Thus, it is clear that most of this activity was added to the reaction buffer, after establishing can be ascribed to other compounds, unknown at that this had negligible effect on the enzymatic re- present. Essential oils can be excluded as active action, to enhance the solubility. Further, as it components in this case, as they evaporate during could not be assumed, that all of the added imper- concentration. atorin was in solution, nor that the initially dis- This is the first study that assesses the effect of solved imperatorin would remain dissolved, the G. sylvaticum on AChE. The active component or reaction mixture containing the highest quantity components are unknown at present. However, it of imperatorin was filtered after the assay and provides some clues to their nature, that an extract measured by HPLC. The results showed, that with 96% ethanol was less active than the extract though up to 274 μg/ml (1.01 mm) was dissolved, with 45% ethanol (results not shown). imperatorin was without any significant effect on Synergistic activity between the extracts of A. the enzymatic reaction in our test system. This is archangelica and G. sylvaticum was demonstrated in contrast with a previous study (Kim et al., 2002). by the isobole method. All tested mixtures had However, in that study, the test system was differ- IC50 values similar to that of the A. archangelica ent, with the sample being incubated with the en- extract, that is much lower than for the G. sylva- zyme for 30 minutes before the reaction buffer ticum extract, even when the latter accounted for was added. more than 84% of the resulting mixture. This ef- Xanthotoxin is a prominent furanocoumarin of fect was most pronounced in mixtures in which the extract, and the second most abundant in the about 80% of the sample is derived from G. sylva- reaction mixture in the assay of the seed extract. ticum. The synergistic effect is mainly visible in It was treated in the same manner as imperatorin, doses at about 2 mg/ml or higher. At lower con- although its solubility was less affected by the de- centrations the dose-response curve lies between μ tergent. The IC50 value was established as 155 g/ those of its constituents. S. Sigurdsson and S. Gudbjarnason · Inhibition of Acetylcholinesterase 693

Akhondzadeh S. and Abbasi S. H. (2006), Herbal medi- tors from the roots of Angelica dahurica. Arch. cine in the treatment of Alzheimer’s disease. Am. J. Pharm. Res. 25, 856Ð859. Alzheimers Dis. Other Demen. 21, 113Ð118. Lee K. H. and Soine T. O. (1969), X. Spectral studies on Ellis J. M. (2005), Cholinesterase inhibitors in the treat- some linear furanocoumarins. J. Pharm. Sci. 58, 681Ð ment of . J. Am. Osteopath. Assoc. 105, 683. 145Ð158. Miyazawa M., Tsukamoto T., Anzai J., and Ishikawa Y. Ellman G. L., Courtney K. D., Andres V., and Feather- (2004), Insecticidal effect of phthalides and furano- stone R. M. (1961), A new and rapid colorimetric de- coumarins from Angelica acutiloba against Drosophila termination of acetylcholinesterase activity. Biochem. melanogaster. J. Agric. Food Chem. 52, 4401Ð4405. Pharmacol. 7,88Ð95. Newall C. A., Andersen L. A., and Phillipson J. D. Hodges J. R. (2006), Alzheimer’s centennial legacy: ori- (1996), Herbal Medicines. A Guide for Health Care gins, landmarks and the current status of knowledge concerning cognitive aspects. Brain 129, 2811Ð2822. Professionals. The Pharmaceutical Press, London. Kamal M. A., Al-Jafari A. A., Yu Q. S., and Greig N. H. Orhan I. and Sener B. (2003), Bioactivity-directed frac- (2006), Kinetic analysis of the inhibition of human bu- tionation of alkaloids from some Amaryllidaceae tyrylcholinesterase with cymserine. Biochim. Biophys. plants and their anticholinesterase activity. Chem. Acta 1760, 200Ð206. Nat. Comp. 39, 383Ð386. Kim D. K., Lim J. P., Yang J. H., Eom D. O., Eun J. S., Williamson E. M. (2001), Synergy and other interactions and Leem K. H. (2002), Acetylcholinesterase inhibi- in phytomedicines. Phytomedicine 8, 401Ð409.